Project Summary For modern flow cytometry, high-throughput multiplexing is extremely important because of the great need in analyzing a large number of biomolecules on and in a single cell. This trend is driven by precision medicine and the need to analyze an ever increasing number of immune-cell subtypes (e.g. in immune-oncology). Highly multiplexed cellular analysis using flow cytometry, however, is challenging. To address this limitation of flow cytometry, we propose to develop a Cyclic Imaging Cytometry (CIC) platform that can be developed to offer a simple automated workflow. The concept behind CIC is straightforward, involving the following steps: 1) Rapid labeling of cells with a large color panel of fluorescent probes against different cellular markers; 2) Perform sensitive and rapid fluorescence imaging; 3) Rapid de-staining of the labeled cells; 4) Iterate steps 1-3 to achieve a large number of imaged biomarkers. While the concept of sequential labeling and de-staining has been explored by many research groups over the years, especially in the context of tissue imaging, this approach thus far has not been able to offer the same level of sensitivity and throughput that is routinely provided by flow cytometry. Recently, we and others have developed fluorescent nanoparticles based on semiconducting polymers called Pdots. The motivation of adapting fluorescent semiconducting polymers into nanoparticle labels stems from a number of favorable characteristics, such as large absorptivity, high quantum yield, fast emission rates, and excellent photostability. The resulting Pdots exhibit extraordinarily high fluorescence brightness, a factor of 102 - 104 higher than conventional dyes, and a factor of 10-103 higher than Qdots depending on the particle size. With the development and availability of Pdots, we believe we can develop CIC to exceed the level of performance offered by flow cytometry. Specifically, the high brightness of Pdots enables high sensitivity imaging and detection of cellular biomarkers, even those present at very low expression levels, and the amplified energy transfer present in Pdots allows the development of a large color panel of Pdots that are both bright with narrow emissions and which can be de-stained efficiently for use in CIC.